CD8+ T cells have been shown to suppress transcription of human immunodeficiency virus type 1 (HIV-1) in a MHC-unrestricted non-cytolytic manner. Yet the identity of the T cell mediated antiviral factor (CAF) remained elusive for last 20 years. Previous studies in the purification of the CAF were largely based on their efforts on an assumption that a soluble factor, such as a cytokine or chemokine, mediates the antiviral activity. We considered an alternative and novel hypothesis that non-cytotoxic HIV-1 suppression is a membrane dependent phenomenon. To prove this hypothesis we have demonstrated a membrane-localized activity mediating HIV-1 transcription suppression that is concomitantly secreted in membrane-bound form through extracellular secretion of 30-100 nanometer sized vesicles known as exosomes. We have been successful in extracting a soluble form of this antiviral activity from purified exosomes using a novel procedure that eliminates serum and cell membrane. Protein mass spectrometric analysis of this serum-free protein fraction with antiviral activity indicated only a limited number of proteins in it. The objective of this study is to conclusively identify the HIV suppressive protein in aqueous soluble extracts of exosomes. Specific aims of the project are: 1) Biochemical isolation of the anti-HIV factor to near purity. A series of biochemical procedures that showed partial purification of antiviral activity in our Preliminary studies will be used in tandem. A reporter gene mediated HIV-1 transcription suppression assay will be used to monitor antiviral activity during purification;2) Identification of a most probable candidate protein/gene sequence for the HIV-1 LTR suppressing factor by a quantitative protein mass spectrometry approach. Quantitative protein mass spectrometric analysis using differential isotope-tagging coupled with multidimensional liquid chromatography (MDLC) will be applied to serum-free protein fraction containing anti-HIV factor;3) Conclusive confirmation of a candidate protein/gene sequence as the LTR promoter suppressive HIV-1 suppressive factor. A combination of RNA-interference, recombinant protein production and/or antibody neutralization of the protein factor will be used to confirm whether a candidate gene sequence is the HIV-1 suppressive factor. . Because of its non-cytolytic mode of action and activity against a diverse range of HIV-1 with different co-receptor properties, the identification of the suppressive factor has a profound effect on therapy and natural history studies of HIV-1 infection. PUBLIC HEALTH RELEVANCE: CD8+ T cells from HIV-1-infected subjects can suppress Human Immunodeficiency Virus type 1 (HIV-1) replication. Identity of this antiviral factor remained elusive for last 20 years. We have recently identified a potent membrane-bound HIV-1 suppressing activity that is secreted from transformed CD8+ T cells as 30-100 nm sized endosome-derived vesicles termed exosomes. We have been successful in extracting a soluble form of this antiviral activity from purified exosomes using a novel procedure that eliminates serum and cell membrane. The objective of the proposed study is to use a combined biochemical and proteomic approach to conclusively identify the HIV-1 suppressive factor from the exosome-derived soluble factor.